The IMViC Tests
The IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae). These
are the Indole test (tryptone broth), the Methyl Red and Voges-Proskauer tests (MRVP
broth) and the Citrate test (Citrate agar slants). For these IMViC tests use the enterics
E. coli and Enterobacter. Work in groups of 4-5 students.
The significance of these tests is that when testing drinking water for the presence of the
sewage indicator E. coli, one must be able to rule out Enterobacter aerogenes.
E. aerogenes is not always associated with sewage, and its presence in water would not
necessarily indicate sewage contamination.
CONTROLS
Escherica. coli
Indole
+
Enterobacter. aerogenes
-
Methyl Red
+
-
VP
-
Citrate
-
+
+
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1. Indole test (tryptone broth)
Procedure: Inoculate a loopful of bacteria into a tryptone broth. Incubate 48 hours.
Description: Tryptophan hydrolysis -Some bacteria split tryptophan into
indole and pyruvic acid using the hydrolase called tryptophanase. Indole
can be detected with Kovac's reagent (Indole reagent). This test is very
important in differentiating E. coli (indole positive) from some closely
related enteric bacteria. It also differentiates Proteus mirabilis (indole
negative) from all other Proteus species (indole positive). Tryptone broth
is used for this test as it contains a large amount of tryptophan.
Interpretation: After incubation: The broth must be turbid. A clear broth
indicates that your organism did not grow and cannot be tested. Add a few
drops of Indole reagent to the broth culture (tryptone broth). DO NOT
SHAKE THE TUBE. A positive result has a red layer at the top. A
negative result has a yellow or brown layer.
2. Methyl Red test (MRVP broth)
Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.
Description: Mixed acid fermentation - Many gram-negative intestinal
bacteria can be differentiated based on the products produced when they
ferment the glucose in MR-VP medium. Escherichia, Salmonella, and
Proteus ferment glucose to produce lactic, acetic, succinic, and formic
acids and CO2, H2, and ethanol. The large amounts of acids produced
lowers the pH of the medium - Methyl red (a pH indicator) will turn red
when added to the medium if the organism was a mixed acid fermenter.
Many of these organisms also produce gas.
Interpretation: After incubation: The broth must be turbid. A clear broth
indicates that your organism did not grow and cannot be tested. Remove 1
ml of broth and place into a sterile tube before performing the methyl red
test if you are going to use the same broth for the VP test. Add 3-4 drops
of methyl red to the original broth. DO NOT SHAKE THE TUBE. A
positive result has a distinct red layer at the top of the broth. A negative
result has a yellow layer.
3. Voges-Proskauer test
Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.
Description: Organisms that are negative in the methyl red test may be
producing 2, 3 butanediol and ethanol instead of acids. These non-acid
products do not lower the pH as much as acids do. Enterobacter, Serratia
and some species of Bacillus produce these substances. There is no
satisfactory test for determining production of 2, 3 butanediol. A precursor
of 2,3 butanediol called acetoin can be detected with Barritt's reagent.
Interpretation: After incubation: Read the VP test when you have good
turbidity. A clear broth indicates that your organism did not grow and
cannot be tested. Barritt's reagent A (VP A) contains naphthol and Barritt's
B (VP B) contains KOH. Test 1 ml of your culture from the MRVP broth.
If you have already conducted the methyl red test, you should have
already placed 1 ml of untested broth in a sterile tube. If you haven’t done
this, do so now. Add the entire contents of the VP A reagent (15 drops)
and 5 drops of the VP B reagent to the 1 ml of your broth culture. SHAKE
WELL. This reaction will take a few minutes before you will see a color
change. SHAKE the tube every few minutes for best results. With a
positive reaction the medium will change to pink or red indicating that
acetoin is present. With a negative reaction the broth will not change color
or will be copper colored. Wait at least 15 minutes for color to develop
before calling the test negative.
4. Citrate test (Simmon's Citrate slant)
Procedure: Streak a loopful of bacteria onto a citrate agar slant, do not stab
the butt. Incubate 24 to 48 hours, longer for Bacillus species. Incubate
with a loose cap.
Description: Simmon's citrate agar tests for the ability of an organism to
use citrate as its sole source of carbon. This media contains a pH indicator
called bromthymol blue. The agar media changes from green to blue at an
alkaline pH.
Interpretation: After incubation: A positive reaction is indicated by a
slant with a Prussian blue color. A negative slant will have no growth of
bacteria and will remain green.
5. Other media: (not part of the IMViC test)
Eosine Methylene Blue (EMB) Agar
Procedure: Streak for isolation.
Description: A selective medium for gram-negative bacteria. Levine’s
EMB agar contains methylene blue, which inhibits gram-positive bacteria.
Differential for enterics: will differentiate lactose fermenters from
nonfermenters.
Interpretation: After incubation: Lactose nonfermenters will have cream
colored colonies. Lactose fermenters will have pinkish colonies,
sometimes with dark centers. E. coli often has a greenish metallic sheen.
Eosine Methylene Blue (EMB) Agar
This media selects for gram-negative
organisms.
Lactose fermenters can be differentiated from
nonfermenters by the color of the colonies.
Nonfermenters have a creamish white colony.
Fermenters have pink colonies often with dark
centers. Escherichiacoli often has a metallic
green sheen, as seen on this Plate.
This is a lactose fermenter that does not produce
the metallic green sheen.
IMViC Tests Results
Indole Test
Bacteria were grown in tryptone
broth.
The uninoculated tube is on the left.
The positive indole test is in the
center.
The negative indole test is on the
right.
Methyl Red (MR) Test
Bacteria were grown in MRVP
broth.
The uninoculated tube is on the left.
The positive MR test is in the
center.
The negative MR test is on the
right.
Voges-Praskauer (VP) Test
Bacteria were grown in MRVP
broth.
The uninoculated tube is on the left.
The positive VP test in in the center.
The negative VP test is on the right.
Citrate test
Bacteria were grown on Citrate
Agar slants.
The uninoculated tube is on the left.
The positive Citrate test in in the
center.
The negative Citrate test is on the
right.
Motility Test
The two tubes on the left
contain a nonmotile bacterial
species. Notice the clearly
visible line of growth (streak
line).
The two tubes on the right
contain a motile bacterial
species. Notice the cloudy
media and the less distinct line
of growth.